Calnexin Associates with Monomeric and Oligomeric (Disulfide-linked) CD3δ Proteins in Murine T Lymphocytes*

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic αβ heterodimers noncovalently associated with monomeric CD3γ,δ,ε proteins and disulfide-linked ζζ homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4+CD8+ thymocytes and splenic T cells. These studies demonstrate that CD3δ proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3ε subunits in CD4+CD8+ thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3δ proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660–9665), we found that glucose residues were not invariably removed from CD3δ glycoproteins prior to their assembly with CD3ε subunits in CD4+CD8+thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3δ proteins in murine T cells. The data in the current report demonstrate that CD3δ proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4+CD8+ thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3δ proteins is a function of their oxidation state.

In general, the surface expression of TCR proteins is tightly associated with their assembly status (1,3). Individual, unas-sembled TCR proteins and partially assembled TCR complexes containing two or three TCR subunits do not effectively exit the ER. Both incomplete ␣␤␦⑀␥⑀ and complete ␣␤␦⑀␥⑀ TCR egress from the ER to the Golgi complex; however, only complete TCR complexes are efficiently transported to the cell surface (1)(2)(3). Unlike mature CD4 ϩ and CD8 ϩ (single positive) T cells, which fundamentally express complete ␣␤␦⑀␥⑀ TCR (3), immature CD4 ϩ CD8 ϩ (double-positive) thymocytes express both complete TCR and partial complexes of CD3␦⑀, CD3␥⑀ proteins associated with calnexin (4 -6), referred to as clonotypic independent complexes (4,5). The molecular basis for clonotypic independent complex expression is unknown but is postulated to result from inefficient ER retention mechanisms in CD4 ϩ CD8 ϩ thymocytes that do not persist in mature T cells (5,7).
Previous studies by Jin et al. reported that a small subfraction of CD3⑀ proteins exists as disulfide-linked dimers in human T lymphocytes (8), which assemble with TCR␤ subunits; CD3⑀ dimers were likewise observed to be present in murine thymocytes, although their assembly status was not evaluated (8). Disulfide-linked heterodimers of CD3␥-⑀ proteins have also been described in REX variant human T cell lines, which fail to express TCR␣ or TCR␣,␤ molecules (9). More recently, Huppa and Ploegh demonstrated that human CD3⑀ molecules translated in vitro in the absence of other TCR proteins have a tendency to form disulfide-linked homooligomers, which assemble with the molecular chaperone calnexin (10). Cotranslation of CD3␦ or CD3␥ molecules was sufficient to maintain CD3⑀ proteins in a principally monomeric phase, however, suggesting that CD3␦ and CD3␥ may guide the folding of CD3⑀ proteins during the initial stages of their biosynthesis (10). In the current study we evaluated the disulfide linkage status of newly synthesized TCR proteins in murine CD4 ϩ CD8 ϩ thymocytes and splenic T cells. These studies show that newly synthesized CD3␦ proteins exist as both monomeric and disulfidelinked molecules that differentially assemble with CD3⑀ molecules in CD4 ϩ CD8 ϩ thymocytes and splenic T cells. In addition, these data document that calnexin associates with both monomeric and oligomeric (disulfide-linked) CD3␦ proteins in murine T lymphocytes.

EXPERIMENTAL PROCEDURES
Animals, Cell Preparation, and Reagents-C57BL/6 (B6) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). CD4 ϩ CD8 ϩ thymocytes were isolated by their adherence to plastic plates coated with anti-CD8 mAb (83-12-5) and were typically Ͼ95% CD4 ϩ CD8 ϩ as described (3). Splenic T cells were purified by incubation of single cell suspensions of spleen cells on tissue culture plates coated with rabbit anti-mouse immunoglobulin, (Organo-Technika-Cappel, Malvern, PA) for 60 min at 37°C, followed by isolation of nonadherent cells. The resultant cell populations were typically 80 -85% CD3 ϩ as determined by flow cytometry analysis.
Antibodies-The following mAb were used in this study: 145-2C11, specific for CD3⑀ proteins associated with CD3␦ or CD3␥ chains (11); HMT 3.1, which recognizes CD3⑀ proteins irrespective of their assembly * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disulfide-linked 26-kDa Proteins Differentially Associate with TCR Subunits in CD4 ϩ CD8 ϩ Thymocytes and Splenic T Cells-As shown in
CD3␦ Proteins Are Assembled into Disulfide-linked Dimers in Murine T Cells-Because their molecular mass is similar to that of CD3 components, we reasoned that disulfide-linked 26-kDa proteins might represent newly synthesized CD3␦ proteins, CD3⑀ proteins, or both. To determine whether CD3␦,⑀ proteins were assembled into disulfide-linked dimers in CD4 ϩ CD8 ϩ thymocytes, anti-CD3␦ precipitates of CD4 ϩ CD8 ϩ thymocytes were analyzed on two-dimensional NR ϫ R gels and immunoblotted with antiserum specific for CD3␦ and CD3⑀ molecules. As shown in Fig. 3, CD3␦ proteins existed as both monomeric and disulfide-linked molecules in CD4 ϩ CD8 ϩ thymocytes (Fig. 3, top panel). In contrast, CD3⑀ proteins were present exclusively as nondisulfide-linked monomers (Fig. 3,  bottom panel). Identical results were obtained in immunoblot experiments of anti-CD3⑀ precipitates of CD4 ϩ CD8 ϩ thymocytes (data not shown). These results show that CD3␦ proteins exist as both monomeric and disulfide-linked molecules in CD4 ϩ CD8 ϩ thymocytes.
CD3␦ and CD3⑀ proteins are easily distinguished from each other in that CD3␦ is post-translationally modified by the addition of three N-linked oligosaccharide chains unlike CD3⑀, which does not contain N-glycans (1, 17). To confirm that CD3␦ glycoproteins were disulfide-linked in murine T cells, digitonin lysates of radiolabeled CD4 ϩ CD8 ϩ thymocytes were immunoprecipitated with anti-CD3␦ Ab, CD3␦ precipitates were boiled in SDS to release bound material, and CD3␦ proteins were recaptured by precipitation with anti-CD3␦ Ab. Precipitates were digested with Endo H glycosidase (specific for cleavage of immature N-linked glycans) and analyzed on one-dimensional SDS-PAGE gels under nonreducing conditions. Most CD3␦ proteins radiolabeled during a 30-min pulse period migrated as monomeric 26-kDa proteins (Fig. 4, first lane), which fell to 17 kDa following removal of N-linked glycan chains, as expected (Fig. 4, second lane). Importantly, these data show that remaining CD3␦ molecules existed as disulfide-linked proteins that migrated at approximately 52 kDa in mock treated samples (Fig. 4, first lane) and at 34 kDa following glycosidase digestion (Fig. 4, second lane); these results were confirmed by immunoblotting experiments using anti-CD3␦ Ab (data not shown). These data are consistent with the assembly of CD3␦ glycoproteins into disulfide-linked dimers that are composed of CD3␦ proteins linked to itself (CD3␦-␦) or to another molecule of similar size (CD3␦-x), which like CD3␦, must also contain Nglycans as the magnitude of decrease in molecular mass following deglycosylation is greater than would be expected for CD3␦ associated with a nonglycosylated protein.
Glc Trimming and Calnexin Assembly of CD3␦ Molecules in CD4 ϩ CD8 ϩ Thymocytes-Immature N-glycan chains on newly synthesized glycoproteins having the structure Glc 3 Man 9 GlcNAc 2 are initially processed by the sequential action of glucosidase I and glucosidase II enzymes in the ER, creating monoglucosylated Glc 1 Man 9 GlcNAc 2 glycans important for interaction with the lectin-like chaperone calnexin (18)(19)(20)(21)(22). Calnexin is proposed to function in the quality control of folding and assembly of numerous newly synthesized glycoproteins, including TCR␣,␤ and CD3␦,␥ subunits (23)(24)(25). Previous studies on the processing of TCR glycoproteins in splenic T cells show that Glc residues are removed from newly synthesized CD3␦ molecules prior to their assembly with other TCR subunits and that calnexin associates exclusively with unassembled, "free" CD3␦ proteins containing incompletely trimmed glycan chains (24). To determine whether CD3␦ proteins were similarly processed in CD4 ϩ CD8 ϩ thymocytes, cells were pulse-labeled with [ 35 S]methionine for 30 min and solubilized in 1% digitonin, and lysates were immunoprecipitated with anti-CD3␦ Ab to purify total CD3␦ proteins; alternatively, lysates were sequentially immunoprecipitated with anti-TCR␤ mAb to isolate CD3␦ proteins assembled into complete ␣␤␦⑀␥⑀ and incomplete ␣␤␦⑀␥⑀

FIG. 3. CD3␦ proteins are assembled into disulfide-linked dimers in murine T cells.
Anti-CD3␦ immunoprecipitates of digitonin lysates of CD4 ϩ CD8 ϩ thymocytes were analyzed on two-dimensional NR ϫ R SDS-PAGE gels and immunoblotted with anti-CD3␦ or anti-CD3⑀ Ab as indicated. The positions of monomeric and disulfide-linked CD3␦ proteins are indicated. Note that anti-CD3⑀ proteins existed exclusively as nondisulfide-linked, monomeric proteins in these studies. TCR complexes, followed by precipitation with anti-CD3⑀ mAb to capture CD3␦ chains present in partial complexes of CD3␦⑀ components and finally precipitation with anti-CD3␦ Ab to purify remaining unassembled, free CD3␦ chains. Precipitates were boiled in SDS to release bound material, CD3␦ proteins were specifically recaptured with anti-CD3␦ Ab, and recapture precipitates were digested with JB and EH glycosidases. JB digestion is useful for evaluating the Glc trimming status of newly synthesized glycoproteins because it removes eight mannoses from fully trimmed Nglycan chains devoid of Glc residues (Man 8-9 GlcNAc 2 ) but only five mannoses from incompletely trimmed N-glycans containing one to three Glc saccharides (Glc 1-3 Man 8-9 GlcNAc 2 ) (19,24). In contrast, EH removes all but a single GlcNAc from N-glycan chains irrespective of their Glc content (26). Similar to what was previously observed in splenic T cells (24), CD3␦ proteins synthesized in CD4 ϩ CD8 ϩ thymocytes existed in four major glycoforms (A-D), indicative of CD3␦ proteins containing three (A), two (B), one (C), and zero (D) incompletely trimmed glycan chains, respectively (Fig.  5A, left-hand side). Interestingly, however, unlike splenic T cells, CD3␦ proteins containing incompletely trimmed N-glycans in CD4 ϩ CD8 ϩ thymocytes were present as both free, unassembled chains and as assembled molecules associated with CD3⑀ proteins (Fig. 5, A and B). In contrast, CD3␦ proteins associated with TCR␤ were totally devoid of Glc residues as shown by their complete sensitivity to JB digestion (Fig. 5, A and B). Taken together, these data show that CD3␦ glycoforms are similarly generated in immature CD4 ϩ CD8 ϩ thymocytes and splenic T cells and that CD3␦ proteins containing incompletely trimmed N-glycans exist as both free and assembled TCR subunits in CD4 ϩ CD8 ϩ thymocytes.
Next, the assembly of newly synthesized CD3␦ proteins with the molecular chaperone calnexin was examined. Metabolically labeled CD3␦ proteins coprecipitated with calnexin in CD4 ϩ CD8 ϩ thymocytes that, as expected, contained incompletely trimmed glycan chains that were partially resistant to JB digestion (Fig. 5A, middle lanes). As similarly noted for other T cell types (25), CD3␦ proteins synthesized in CD4 ϩ CD8 ϩ thymocytes did not associate with the calnexinrelated molecule, calreticulin (data not shown). To determine the disulfide linkage status of CD3␦ proteins associated with calnexin, digitonin lysates of radiolabeled CD4 ϩ CD8 ϩ thymocytes were immunoprecipitated with anti-calnexin Ab, precipitates were boiled in 1% SDS to release bound material, and CD3␦ proteins were specifically recaptured with anti-CD3␦ Ab. Analysis of recapture precipitates on two-dimensional NR ϫ R SDS-PAGE gels showed that both monomeric and disulfidelinked CD3␦ proteins were assembled with calnexin in CD4 ϩ CD8 ϩ thymocytes (Fig. 6); similar results were observed in splenic T cells (data not shown). These studies show that both monomeric and disulfide-linked CD3␦ proteins associate with calnexin in murine T cells. DISCUSSION In the current report we evaluated the disulfide linkage status of newly synthesized TCR proteins in CD4 ϩ CD8 ϩ thymocytes and splenic T cells. These studies show that: (i) CD3␦ proteins exist as both monomeric and oligomeric (disulfidelinked) species in murine T cells; (ii) disulfide-linked CD3␦ proteins differentially assemble with CD3⑀ and TCR␤ subunits FIG. 4. A subset of newly synthesized CD3␦ glycoproteins exists as disulfide-linked dimers in CD4 ؉ CD8 ؉ thymocytes. Digitonin lysates of [ 35 S]methionine-radiolabeled CD4 ϩ CD8 ϩ thymocytes were immunoprecipitated with anti-CD3␦ Ab, precipitates were boiled in 1% SDS to release bound material, and CD3␦ proteins were specifically recaptured by precipitation with anti-CD3␦ Ab. Recapture precipitates were digested with EH glycosidases as indicated and analyzed on 13% SDS-PAGE gels under nonreducing conditions. The positions of monomeric and disulfide-linked CD3␦ proteins are marked. An asterisk indicates a nonglycosylated molecule that nonspecifically coprecipitates with CD3␦ proteins, believed to be actin; this molecule is not disulfidelinked to CD3␦ proteins as determined by analysis on two-dimensional nonreducing ϫ reducing gels (see Fig. 6).
FIG. 5. Glc trimming and TCR assembly of CD3␦ glycoproteins in CD4 ؉ CD8 ؉ thymocytes. A, digitonin lysates of [ 35 S]methionineradiolabeled CD4 ϩ CD8 ϩ thymocytes were immunoprecipitated with anti-CD3␦ Ab or anti-calnexin Ab or were sequentially immunoprecipitated with anti-TCR␤ mAb, followed by anti-CD3⑀ mAb, and finally were immunoprecipitated with anti-CD3␦ Ab. Precipitated material was released by boiling in SDS and CD3␦ proteins specifically recaptured by precipitation with anti-CD3␦ Ab; recapture precipitates were digested with JB and EH glycosidases as indicated. The positions of CD3␦ glycoforms (A-D) and Endo-H-sensitive, deglycosylated CD3␦ proteins (CD3␦ EH S ) are marked. B, same as in A except that CD4 ϩ CD8 ϩ thymocyte lysates were sequentially immunoprecipitated with anti-TCR␤ mAb, followed by immunoprecipitation with anti-CD3⑀ mAb and finally with anti-CD3␦ Ab.
in CD4 ϩ CD8 ϩ thymocytes and splenic T cells; (iii) unlike CD3␦ processing in splenic T cells, Glc residues are not invariably removed from CD3␦ glycoproteins prior to their assembly with CD3⑀ chains in CD4 ϩ CD8 ϩ thymocytes; and (iv) calnexin associates with both monomeric and disulfide-linked CD3␦ proteins in murine T cells.
Previous studies by Jin et al. reported that a fraction of CD3⑀ proteins exists as disulfide-linked dimers in murine T cells, including thymocytes (8). The data in the current study show that CD3␦ proteins were present as disulfide-linked dimers in both CD4 ϩ CD8 ϩ thymocytes and splenic T cells, but no evidence was found for disulfide linkage of CD3⑀ molecules in either cell type. The reason for these apparent discrepancies are unclear but may result from the fact that identification of disulfide-linked CD3⑀ proteins in previous studies relied on their detection by rabbit antiserum directed against murine CD3⑀ (8), which may detect unique epitopes not recognized by the anti-CD3⑀ mAbs used in our study. Regardless, the current study clearly demonstrates that CD3␦ glycoproteins were assembled into disulfide-linked dimers in murine T lymphocytes using several different approaches, including immunoblotting, immunoprecipitation/release/recapture experiments, and glycosidase digestion studies. Our results are consistent with the assembly of CD3␦ chains into homodimers containing CD3␦ proteins disulfide-linked to itself or heterodimers of CD3␦ proteins disulfide-bridged to another molecular of similar molecular mass and carbohydrate content.
Huppa and Ploegh have recently shown that human CD3⑀ molecules translated in vitro in the absence of other TCR proteins have a tendency to form disulfide-linked homooligomers, which assemble with calnexin (10). The data in the current report importantly extend these studies by showing that murine CD3␦ proteins synthesized in intact cells in the presence of other TCR proteins may also exist as disulfidelinked molecules that can assemble with calnexin. Conceivably, disulfide-linked CD3␦ proteins in CD4 ϩ CD8 ϩ thymocytes and splenic T cells may represent CD3␦ molecules that are synthesized in excess of other TCR subunits that rapidly dimerize and bind to calnexin. Curiously, however, disulfide-linked CD3␦ proteins were not assembled with other TCR molecules in splenic T cells but were (noncovalently) associated with CD3⑀ proteins in CD4 ϩ CD8 ϩ thymocytes. Although the significance of these findings is currently unclear, it is interesting to note that these results parallel the differential assembly of Glccontaining CD3␦ proteins with CD3⑀ molecules in CD4 ϩ CD8 ϩ thymocytes and splenic T cells. The molecular basis for the differential assembly of incompletely trimmed CD3␦ chains with CD3⑀ proteins in CD4 ϩ CD8 ϩ thymocytes and splenic T cells is unknown but may be influenced by the dissimilar stability of TCR␣ proteins in these two cell types (3,17). We have previously noted that CD3␦ proteins synthesized in T hybridoma cells under conditions of impaired glucosidase activity (which destabilizes TCR␣ molecules) show increased assembly with CD3⑀ subunits, similar to what is naturally observed in CD4 ϩ CD8 ϩ thymocytes (3). Thus, it is possible that TCR␣ association with CD3␦ or CD3⑀ proteins retards assembly of disulfide-linked CD3␦ proteins with CD3⑀ molecules, although such ␣␦,␣⑀ intermediates remain to be directly demonstrated in primary murine T cells (3). In both CD4 ϩ CD8 ϩ thymocytes and splenic T cells, dimeric CD3␦ proteins were precluded from incorporation into complete TCR complexes as evidenced by their failure to coprecipitate with TCR␤, , and CD3␥ proteins (this study), 2 indicating that quality control mechanisms exist in both cell types that control assembly of dimeric CD3␦ proteins into higher ordered TCR complexes.
Finally, our results that newly synthesized CD3␦ chains bearing incompletely trimmed oligosaccharides were assembled with CD3⑀ subunits in CD4 ϩ CD8 ϩ thymocytes are in agreement with previous reports that Glc-containing CD3␦ proteins are expressed on the surfaces of immature thymocytes in association with CD3⑀ molecules (5,7). Importantly, however, the data in the current study provide the first assessment of the efficiency of Glc removal from newly synthesized CD3␦ proteins in CD4 ϩ CD8 ϩ thymocytes and show that CD3␦ glycoforms are effectively generated in CD4 ϩ CD8 ϩ thymocytes as in splenic T cells (24).